Circulating bacterial peptides and linked metabolomic signatures are indicative of early mortality in pediatric cirrhosis.

BACKGROUND: Patients with pediatric cirrhosis-sepsis (PC-S) attain early mortality. Plasma bacterial composition, the cognate metabolites, and their contribution to the deterioration of patients with PC-S to early mortality are unknown. We aimed to delineate the plasma metaproteome-metabolome landscape and identify molecular indicators capable of segregating patients with PC-S predisposed to early mortality in plasma, and we further validated the selected metabolite panel in paired 1-drop blood samples using untargeted metaproteomics-metabolomics by UHPLC-HRMS followed by validation using machine-learning algorithms. METHODS: We enrolled 160 patients with liver diseases (cirrhosis-sepsis/nonsepsis [n=110] and noncirrhosis [n=50]) and performed untargeted metaproteomics-metabolomics on a training cohort of 110 patients (Cirrhosis-Sepsis/Nonsepsis, n=70 and noncirrhosis, n=40). The candidate predictors were validated on 2 test cohorts-T1 (plasma test cohort) and T2 (1-drop blood test cohort). Both T1 and T2 had 120 patients each, of which 70 were from the training cohort. RESULTS: Increased levels of tryptophan metabolites and Salmonella enterica and Escherichia coli-associated peptides segregated patients with cirrhosis. Increased levels of deoxyribose-1-phosphate, N5-citryl-d-ornithine, and Herbinix hemicellulolytic and Leifsonia xyli segregated patients with PC-S. MMCN-based integration analysis of WMCNA-WMpCNA identified key microbial-metabolic modules linked to PC-S nonsurvivors. Increased Indican, Staphylobillin, glucose-6-phosphate, 2-octenoylcarnitine, palmitic acid, and guanidoacetic acid along with L. xyli, Mycoplasma genitalium, and Hungateiclostridium thermocellum segregated PC-S nonsurvivors and superseded the liver disease severity indices with high accuracy, sensitivity, and specificity for mortality prediction using random forest machine-learning algorithm. CONCLUSIONS: Our study reveals a novel metabolite signature panel capable of segregating patients with PC-S predisposed to early mortality using as low as 1-drop blood.

Comparative metabolome analysis reveals higher potential of haemoperfusion adsorption in providing favourable outcome in ACLF patients.

BACKGROUND AND AIMS: Acute-on-chronic liver failure (ACLF) is a serious illness associated with altered metabolome, organ failure and high mortality. Need for therapies to improve the metabolic milieu and support liver regeneration are urgently needed. METHODS: We investigated the ability of haemoperfusion adsorption (HA) and therapeutic plasma exchange (TPE) in improving the metabolic profile and survival in ACLF patients. Altogether, 45 ACLF patients were randomized into three groups: standard medical therapy (SMT), HA and TPE groups. Plasma metabolomics was performed at baseline, post-HA and TPE sessions on days 7 and 14 using high-resolution mass spectrometry. RESULTS: The baseline clinical/metabolic profiles of study groups were comparable. We identified 477 metabolites. Of these, 256 metabolites were significantly altered post 7 days of HA therapy (p < .05, FC > 1.5) and significantly reduced metabolites linked to purine (12 metabolites), tryptophan (7 metabolites), primary bile acid (6 metabolites) and arginine-proline metabolism (6 metabolites) and microbial metabolism respectively (p < .05). Metabolites linked to taurine-hypotaurine and histidine metabolism were reduced and temporal increase in metabolites linked to phenylalanine and tryptophan metabolism was observed post-TPE therapy (p < .05). Finally, weighted metabolite correlation network analysis (WMCNA) along with inter/intragroup analysis confirmed significant reduction in inflammatory (tryptophan, arachidonic acid and bile acid metabolism) and secondary energy metabolic pathways post-HA therapy compared to TPE and SMT (p < .05). Higher baseline plasma level of 11-deoxycorticosterone (C03205; AUROC > 0.90, HR > 3.2) correlated with severity (r2 > 0.5, p < .05) and mortality (log-rank-p < .05). Notably, 51 of the 64 metabolite signatures (ACLF non-survivor) were reversed post-HA treatment compared to TPE and SMT(p < .05). CONCLUSION: HA more potentially (~80%) improves plasma milieu compared to TPE and SMT. High baseline plasma 11-deoxycorticosterone level correlates with early mortality in ACLF patients.

Modulation of CD8+T cells, NK cells and Th1cytokines by metabolic milieu in decline of HBV-viremia in pregnant women treated with tenofovir-disoproxil from second trimester of pregnancy.

High HBV DNA levels predispose to mother to child transmission (MTCT) of HBV. Early nucleotide analogue (NA) therapy can reduce HBV DNA and minimize MTCT. We analysed immune-metabolic profile in pregnant mothers who received NA from 2nd trimester compared with untreated mothers. In 2nd trimester, there was no difference in immune profiles between Gr.1 and Gr.2 but high viral load women had downregulated pyruvate, NAD+ metabolism but in 3rd trimester, Gr.1 had significant reduction in HBV-DNA, upregulated pyruvate and NAD with increased IFN-2αA, CD8Tcells, NK cells and decreased Tregs, IL15, IL18, IL29, TGFß3 compared to Gr.2. In Gr.1, three eAg-ve women showed undetectable DNA and HBsAg. At delivery, Gr.1 showed no MTCT, with undetectable HBV DNA, HBsAg, high CD8 and NK cells in two women. We conclude, that starting NA from second trimester, reduces HBV load and MTCT, modulates NAD, induces immunity and suggest use of NA in early gestation in future trials.

A mitochondrial quality control mechanism reverses the phagosome maturation arrest caused by Mycobacterium tuberculosis.

Phagosome maturation arrest (PMA) imposed by Mycobacterium tuberculosis ( Mtb ) is a classic tool that helps Mtb evade macrophage anti-bacterial responses. The exclusion of RAB7, a small GTPase, from Mtb -phagosomes underscores PMA. Here we report an unexpected mechanism that triggers crosstalk between the mitochondrial quality control (MQC) and the phagosome maturation pathways that reverses the PMA. CRISPR-mediated p62/SQSTM1 depletion ( p62 KD ) blocks mitophagy flux without impacting mitochondrial quality. In p62 KD cells, Mtb growth and survival are diminished, mainly through witnessing an increasingly oxidative environment and increased lysosomal targeting. The lysosomal targeting of Mtb is facilitated by enhanced TOM20 + mitochondria-derived vesicles (MDVs) biogenesis, a key MQC mechanism. In p62 KD cells, TOM20 + -MDVs biogenesis is MIRO1/MIRO2-dependent and delivered to lysosomes for degradation in a RAB7-dependent manner. Upon infection in p62 KD cells, TOM20 + -MDVs get extensively targeted to Mtb -phagosomes, inadvertently facilitating RAB7 recruitment, PMA reversal and lysosomal targeting of Mtb . Triggering MQC collapse in p62 KD cells further diminishes Mtb survival signifying cooperation between redox- and lysosome-mediated mechanisms. The MQC-anti-bacterial pathway crosstalk could be exploited for host-directed anti-tuberculosis therapies.

Ocimum sanctum Alters the Lipid Landscape of the Brain Cortex and Plasma to Ameliorate the Effect of Photothrombotic Stroke in a Mouse Model.

Stroke-like injuries in the brain result in not only cell death at the site of the injury but also other detrimental structural and molecular changes in regions around the stroke. A stroke-induced alteration in the lipid profile interferes with neuronal functions such as neurotransmission. Preventing these unfavorable changes is important for recovery. Ocimum sanctum (Tulsi extract) is known to have anti-inflammatory and neuroprotective properties. It is possible that Tulsi imparts a neuroprotective effect through the lipophilic transfer of active ingredients into the brain. Hence, we examined alterations in the lipid profile in the cerebral cortex as well as the plasma of mice with a photothrombotic-ischemic-stroke-like injury following the administration of a Tulsi extract. It is also possible that the lipids present in the Tulsi extract could contribute to the lipophilic transfer of active ingredients into the brain. Therefore, to identify the major lipid species in the Tulsi extract, we performed metabolomic and untargeted lipidomic analyses on the Tulsi extract. The presence of 39 molecular lipid species was detected in the Tulsi extract. We then examined the effect of a treatment using the Tulsi extract on the untargeted lipidomic profile of the brain and plasma following photothrombotic ischemic stroke in a mouse model. Mice of the C57Bl/6j strain, aged 2-3 months, were randomly divided into four groups: (i) Sham, (ii) Lesion, (iii) Lesion plus Tulsi, and (iv) Lesion plus Ibuprofen. The cerebral cortex of the lesioned hemisphere of the brain and plasma samples were collected for untargeted lipidomic profiling using a Q-Exactive Mass Spectrometer. Our results documented significant alterations in major lipid groups, including PE, PC, neutral glycerolipids, PS, and P-glycerol, in the brain and plasma samples from the photothrombotic stroke mice following their treatment with Tulsi. Upon further comparison between the different study groups of mice, levels of MGDG (36:4), which may assist in recovery, were found to be increased in the brain cortexes of the mice treated with Tulsi when compared to the other groups (p < 0.05). Lipid species such as PS, PE, LPG, and PI were commonly altered in the Sham and Lesion plus Tulsi groups. The brain samples from the Sham group were specifically enriched in many species of glycerol lipids and had reduced PE species, while their plasma samples showed altered PE and PS species when compared to the Lesion group. LPC (16:1) was found in the Tulsi extract and was significantly increased in the brains of the PTL-plus-Tulsi-treated group. Our results suggest that the neuroprotective effect of Tulsi on cerebral ischemia may be partially associated with its ability to regulate brain and plasma lipids, and these results may help provide critical insights into therapeutic options for cerebral ischemia or brain lesions.

SIRT2 inhibition by AGK2 enhances mycobacteria-specific stem cell memory responses by modulating beta-catenin and glycolysis.

Bacille Calmette-Guerin (BCG) generates limited long-lasting adaptive memory responses leading to short-lived protection against adult pulmonary tuberculosis (TB). Here, we show that host sirtuin 2 (SIRT2) inhibition by AGK2 significantly enhances the BCG vaccine efficacy during primary infection and TB recurrence through enhanced stem cell memory (TSCM) responses. SIRT2 inhibition modulated the proteome landscape of CD4+ T cells affecting pathways involved in cellular metabolism and T-cell differentiation. Precisely, AGK2 treatment enriched the IFNγ-producing TSCM cells by activating ß-catenin and glycolysis. Furthermore, SIRT2 specifically targeted histone H3 and NF-κB p65 to induce proinflammatory responses. Finally, inhibition of the Wnt/ß-catenin pathway abolished the protective effects of AGK2 treatment during BCG vaccination. Taken together, this study provides a direct link between BCG vaccination, epigenetics, and memory immune responses. We identify SIRT2 as a key regulator of memory T cells during BCG vaccination and project SIRT2 inhibitors as potential immunoprophylaxis against TB.

Biomolecular map of albumin identifies signatures of severity and early mortality in acute liver failure.

BACKGROUND & AIMS: Acute liver failure (ALF) is associated with high mortality. Alterations in albumin structure and function have been shown to correlate with outcomes in cirrhosis. We undertook a biomolecular analysis of albumin to determine its correlation with hepatocellular injury and early mortality in ALF. METHODS: Altogether, 225 participants (200 patients with ALF and 25 healthy controls [HC]) were enrolled. Albumin was purified from the baseline plasma of the training cohort (ALF, n = 40; survivors, n = 8; non-survivors, n = 32; and HC, n = 5); analysed for modifications, functionality, and bound multi-omics signatures; and validated in a test cohort (ALF, n = 160; survivors, n = 53; non-survivors, n = 107; and HC, n = 20). RESULTS: In patients with ALF, albumin is more oxidised and glycosylated with a distinct multi-omics profile than that in HC, more so in non-survivors (p <0.05). In non-survivors, albumin was more often bound (p <0.05, false discovery rate <0.01) to proteins associated with inflammation, advanced glycation end product, metabolites linked to arginine, proline metabolism, bile acid, and mitochondrial breakdown products. Increased bacterial taxa (Listeria, Clostridium, etc.) correlated with lipids (triglycerides [4:0/12:0/12:0] and phosphatidylserine [39:0]) and metabolites (porphobilinogen and nicotinic acid) in non-survivors (r2 >0.7). Multi-omics signature-based probability of detection for non-survival was >90% and showed direct correlation with albumin functionality and clinical parameters (r2 >0.85). Probability-of-detection metabolites built on the top five metabolites, namely, nicotinic acid, l-acetyl carnitine, l-carnitine, pregnenolone sulfate, and N-(3-hydroxybutanoyl)-l-homoserine lactone, showed diagnostic accuracy of 98% (AUC 0.98, 95% CI 0.95-1.0) and distinguish patients with ALF predisposed to early mortality (log-rank <0.05). On validation using high-resolution mass spectrometry and five machine learning algorithms in test cohort 1 (plasma and paired one-drop blood), the metabolome panel showed >92% accuracy/sensitivity and specificity for prediction of mortality. CONCLUSIONS: In ALF, albumin is hyperoxidised and substantially dysfunctional. Our study outlines distinct 'albuminome' signatures capable of distinguishing patients with ALF predisposed to early mortality or requiring emergency liver transplantation. IMPACTS AND IMPLICATIONS: Here, we report that the biomolecular map of albumin is distinct and linked to severity and outcome in patients with acute liver failure (ALF). Detailed structural, functional, and albumin-omics analysis in patients with ALF led to the identification and classification of albumin-bound biomolecules, which could segregate patients with ALF predisposed to early mortality. More importantly, we found albumin-bound metabolites indicative of mitochondrial damage and hyperinflammation as a putative indicator of <30-day mortality in patients with ALF. This preclinical study validates the utility of albuminome analysis for understanding the pathophysiology and development of poor outcome indicators in patients with ALF.

Berberine governs NOTCH3/AKT signaling to enrich lung-resident memory T cells during tuberculosis.

Stimulation of naïve T cells during primary infection or vaccination drives the differentiation and expansion of effector and memory T cells that mediate immediate and long-term protection. Despite self-reliant rescue from infection, BCG vaccination, and treatment, long-term memory is rarely established against Mycobacterium tuberculosis (M.tb) resulting in recurrent tuberculosis (TB). Here, we show that berberine (BBR) enhances innate defense mechanisms against M.tb and stimulates the differentiation of Th1/Th17 specific effector memory (TEM), central memory (TCM), and tissue-resident memory (TRM) responses leading to enhanced host protection against drug-sensitive and drug-resistant TB. Through whole proteome analysis of human PBMCs derived from PPD+ healthy individuals, we identify BBR modulated NOTCH3/PTEN/AKT/FOXO1 pathway as the central mechanism of elevated TEM and TRM responses in the human CD4+ T cells. Moreover, BBR-induced glycolysis resulted in enhanced effector functions leading to superior Th1/Th17 responses in human and murine T cells. This regulation of T cell memory by BBR remarkably enhanced the BCG-induced anti-tubercular immunity and lowered the rate of TB recurrence due to relapse and re-infection. These results thus suggest tuning immunological memory as a feasible approach to augment host resistance against TB and unveil BBR as a potential adjunct immunotherapeutic and immunoprophylactic against TB.

Withaferin A Protects against Primary and Recurrent Tuberculosis by Modulating Mycobacterium-Specific Host Immune Responses.

The fate of Mycobacterium tuberculosis infection is governed by immune signaling pathways that can either eliminate the pathogen or result in tuberculosis (TB). Anti-TB therapy (ATT) is extensive and is efficacious only against active, drug-sensitive strains of M. tuberculosis. Due to severe side effects, ATT often causes impairment of host immunity, making it imperative to use novel immunotherapeutics for better clinical outcomes. In this study, we have explored the immunomodulatory potential of withaferin A (WA) as an immunotherapeutic against TB. Here, we demonstrate that WA can constrain intracellular drug-sensitive and -resistant strains of M. tuberculosis by augmenting host immune responses. We also established the potential of WA treatment in conjunction with isoniazid. We show that WA directs the host macrophages toward defensive M1 polarization and enhances TH1 and TH17 immune responses against M. tuberculosis infection. The reduced bacterial burden upon T cell adoptive transfer further corroborated the augmented T cell responses. Interestingly, WA stimulated the generation of T cell memory populations by instigating STAT signaling, thereby reducing the rate of TB recurrence due to reactivation and reinfection. We substantiate the prospects of WA as a potent adjunct immunomodulator that enriches protective memory cells by prompting STAT signaling and improves host defense against M. tuberculosis. IMPORTANCE Despite being extensive, conventional antituberculosis therapy (ATT) is barely proficient in providing sterile immunity to tuberculosis (TB). Failure to constrain the escalating global TB burden due to the emergence of drug-resistant bacterial strains and immune dampening effects of ATT necessitates adjunct immunotherapeutics for better clinical outcomes. We evaluated the prospects of withaferin A (WA), an active constituent of Withania somnifera, as an adjunct immunomodulator against diverse M. tuberculosis strains. WA efficiently restricts the progression of TB by stimulating antimycobacterial host responses, protective immune signaling, and activation of diverse immune cell populations. Protective effects of WA can be attributed to the enrichment of memory T cells by induction of STAT signaling, thereby enhancing resistance to reinfections and reactivation of disease. We ascertained the immunotherapeutic potential of WA in boosting host immune responses against M. tuberculosis.

Metabolomic analysis shows dysregulation in amino acid and NAD+ metabolism in palmitate treated hepatocytes and plasma of non-alcoholic fatty liver disease spectrum.

There is an alarming increase in incidence of fatty liver disease worldwide. The fatty liver disease spectrum disease ranges from simple steatosis (NAFL) to steatohepatitis (NASH) which culminates in cirrhosis and cancer. Altered metabolism is a hallmark feature associated with fatty liver disease and palmitic acid is the most abundant saturated fatty acid, therefore, the aim of this study was to compare metabolic profiles altered in hepatocytes treated with palmitic acid and also the differentially expressed plasma metabolites in spectrum of nonalcoholic fatty liver. The metabolites were analyzed by liquid chromatography-mass spectrometry (LC-MS) platform. Hepatocyte cell lines PH5CH8 and HepG2 cells when treated with 400 µM dose of palmitic acid showed typical features of steatosis. Metabolomic analysis of lipid treated hepatocyte cell lines showed differential changes in phenylalanine and tyrosine pathways, fatty acid metabolism and bile acids. The key metabolites tryptophan, kynurenine and carnitine differed significantly between subjects with NAFL, NASH and those with cirrhosis. As the tryptophan-kynurenine axis is also involved in denovo synthesis of NAD+, we found significant alterations in the NAD+ related metabolites in both palmitic acid treated and also fatty liver disease with cirrhosis. The study underscores the importance of amino acid and NAD+supplementation as promising strategies in fatty liver disorder.

Plasma Proteomic Analysis Identified Proteins Associated with Faulty Neutrophils Functionality in Decompensated Cirrhosis Patients with Sepsis.

Decompensated cirrhosis (DC) is susceptible to infections and sepsis. Neutrophils and monocytes provide the first line of defense to encounter infection. We aimed to evaluate proteins related to neutrophils functionality in sepsis. 70 (DC), 40 with sepsis, 30 without (w/o) sepsis and 15 healthy controls (HC) plasma was analyzed for proteomic analysis, cytokine bead array, endotoxin, cell free DNA and whole blood cells were analyzed for nCD64-mHLADR index, neutrophils-monocytes, functionality and QRT-PCR. nCD64-mHLADR index was significantly increased (p < 0.0001) with decreased HLA-DR expression on total monocytes in sepsis (p = 0.045). Phagocytic activity of both neutrophils and monocytes were significantly decreased in sepsis (p = 0.002 and p = 0.0003). Sepsis plasma stimulated healthy neutrophils, showed significant increase in NETs (neutrophil extracellular traps) and cell free DNA (p = 0.049 and p = 0.04) compared to w/o sepsis and HC. Proteomic analysis revealed upregulated- DNAJC13, TMSB4X, GPI, GSTP1, PNP, ANPEP, COTL1, GCA, APOA1 and PGAM1 while downregulated- AHSG, DEFA1,SERPINA3, MPO, MMRN1and PROS1 proteins (FC > 1.5; p < 0.05) associated to neutrophil activation and autophagy in sepsis. Proteins such as DNAJC13, GPI, GSTP1, PNP, ANPEP, COTL1, PGAM1, PROS1, MPO, SERPINA3 and MMRN1 showed positive correlation with neutrophils activity and number, oxidative burst activity and clinical parameters such as MELD, MELD Na and Bilirubin. Proteomic analysis revealed that faulty functionality of neutrophils may be due to the autophagy proteins i.e., DNAJC13, AHSG, TMSB4X, PROS1 and SERPINA3, which can be used as therapeutic targets in decompensated cirrhosis patients with sepsis.

Downregulation of gamma subunit of TCP1 chaperonin of Leishmania donovani modulates extracellular vesicles-mediated macrophage microbicidal function.

T-complex protein-1 (TCP1) is a group II chaperonin, known to fold various proteins like actin and tubulin. In Leishmania donovani only one subunit that is gamma subunit (LdTCP1γ) has been functionally characterized as a homo-oligomeric complex that exhibits ATP-dependent protein folding. The gene is essential for the survival and infectivity of the parasite. Leishmania parasite releases extracellular vesicles (EVs) containing numerous virulence factors, which play an essential role in parasite pathogenesis and modulate host immune cell signaling. The present study demonstrates that LdTCP1γ is secreted in the EVs and modulates host macrophage functions. EVs isolated from LdTCP1γ single-allele-replacement mutants significantly upregulate the microbicidal function of LPS-induced macrophage as evident by increased levels of proinflammatory cytokines (TNF-α, IL-6), iNOS and NO production. Further, the comparative proteomics of wild-type and single-allele-replacement mutant EVs showed that out of 876 identified proteins, 207 were significantly modulated. Among them, the top 50 modulated and abundantly secreted proteins constitute ∼40% of the total identified protein intensity and include virulence factors such as GP63, peroxiredoxin, enolase, HSP70, elongation factor 2, amastin, eukaryotic translation initiation factor and α-tubulin. The comparative proteomic analysis revealed that the proteome enrichment of the EVs from LdTCP1γ single-allele replacement mutants significantly differs from wild-type EVs, which may be responsible for the altered host microbicidal responses. Thus, our data provide new insight into the role of LdTCP1γ in EVs-mediated host-parasite interactions.

Bile multi-omics analysis classifies lipid species and microbial peptides predictive of carcinoma of gallbladder.

BACKGROUND AND AIMS: Histopathological examination is the gold standard for detection of gallstone (GS) or gallbladder carcinoma (CAGB). Bile concentrated in the gallbladder (GB) is expected to recapitulate metagenomics and molecular changes associated with development of CAGB. APPROACH AND RESULTS: Bile samples were screened for lipidomics and metaproteome (metagenomics) signatures capable of early detection of cancer in GB anomalies. Analysis of the training cohort (n = 87) showed that metastability of bile was reduced in CAGB (p < 0.05). CAGB bile showed significant alteration of lipidome and microbiome as indicated by multivariate partial least squares regression analysis and alpha-diversity and beta-diversity indexes (p < 0.05). Significant reduction of lipid species and increase in bacterial taxa were found to be associated with patients with CAGB, CAGB with GS, and GS (p < 0.05, log fold change >1.5). A multimodular correlation network created using weighted lipid/metaproteomic correlation network analysis showed striking associations between lipid and metaproteomic modules and functionality. CAGB-linked metaproteomic modules/functionality directly correlated with lipid modules, species, clinical parameters, and bile acid profile (p < 0.05). Increased bacterial taxa (Leptospira, Salmonella enterica, Mycoplasma gallisepticum) and their functionality showed a direct correlation with lipid classes such as lysophosphatidylinositol, ceramide 1-phosphate, and lysophosphatidylethanolamine and development of CAGB (r2  > 0.85). Lipid/metaproteomic signature-based probability of detection for CAGB was > 90%, whereas that for GS was > 80% (p < 0.05). Validation of eight lipid species using four machine learning algorithms in two separate cohorts (n = 38; bile [test cohort 1] and paired plasma [test cohort 2]) showed accuracy (99%) and sensitivity/specificity (>98%) for CAGB detection. CONCLUSIONS: Bile samples of patients with CAGB showed significant reduction in lipid species and increase in bacterial taxa. Our study identifies a core set of bile lipidome and metaproteome signatures which may offer universal utility for early diagnosis of CAGB.

Unraveling of interacting protein network of chaperonin TCP1 gamma subunit of Leishmania donovani.

T-complex polypeptide-1 (TCP1) is a group II chaperonin that folds various cellular proteins. About 10% of cytosolic proteins in yeast have been shown to flux through the TCP1 protein complex indicating that it interacts and folds a plethora of substrate proteins that perform essential functions. In Leishmania donovani, the gamma subunit of TCP1 (LdTCP1γ) has been shown to form a homo-oligomeric complex and exhibited ATP-dependent protein folding activity. LdTCP1γ is essential for the growth and infectivity of the parasite. The interacting partners of L. donovani TCP1γ, involved in many cellular processes, are far from being understood. In this study, we utilized co-immunoprecipitation assay coupled with liquid chromatography-mass spectrometry (LC-MS) to unravel protein-protein interaction (PPI) networks of LdTCP1γ in the L. donovani parasite. Label-free quantification (LFQ) proteomic analysis revealed 719 interacting partners of LdTCP1γ. String analysis showed that LdTCP1γ interacts with all subunits of TCP1 complex as well as other proteins belonging to pathways like metabolic process, ribosome, protein folding, sorting, and degradation. Trypanothione reductase, identified as one of the interacting partners, is refolded by LdTCP1γ. In addition, the differential expression of LdTCP1γ modulates the trypanothione reductase activity in L. donovani parasite. The study provides novel insight into the role of LdTCP1γ that will pave the way to better understand parasite biology by identifying the interacting partners of this chaperonin.

Global metabolome profiling of COVID-19 respiratory specimen using high-resolution mass spectrometry (HRMS).

Here we describe a protocol for identifying metabolites in respiratory specimens of patients that are SARS-CoV-2 positive, SARS-CoV-2 negative, or H1N1 positive. This protocol provides step-by-step instructions on sample collection from patients, followed by metabolite extraction. We use ultra-high-pressure liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS) for data acquisition and describe the steps for data analysis. The protocol was standardized with specific customization for SARS-CoV-2-containing respiratory specimens. For complete details on the use and execution of this protocol, please refer to Maras et al. (2021).

Multi-omics analysis of respiratory specimen characterizes baseline molecular determinants associated with SARS-CoV-2 outcome.

Rapid diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection still remains a major challenge. A multi-omic approach was adopted to analyze the respiratory specimens of 20 SARS-CoV-2-positive, 20 negative and 15 H1N1 pdm 2009 positive cases. Increased basal level of MX1 (MX dynamin-like GTPase 1) and WARS (tryptophan-tRNA ligase) correlated with SARS-CoV-2 infection and its outcome. These markers were further validated in 200 suspects. MX1>30pg/ml and WARS>25ng/ml segregated virus positives [AUC = 94% CI: (0.91-0.97)] and severe patients [AUC>0.85%]. Our results documented significant increase in immune activation; metabolic reprograming and decrease in oxygen transport, wound healing and others linked proteins and metabolites in patients with coronavirus disease 2019 (COVID-19). Multi-omics profiling correlated with viremia and segregated asymptomatic patients with COVID-19. Additionally, we identified increased respiratory pathogens (Burkholderiales, Klebsiella pneumonia) and decreased lactobacillus salivarius (FDR<0.05) in COVID-19 specimens. In conclusion, increased basal MX1 and WARS levels correlates with SARS-CoV-2 infection and could aid in the identification of patient's predisposed to higher severity.

Characterization of a Bowman-Birk type trypsin inhibitor purified from seeds of Solanum surattense.

A Bowman-Birk type trypsin inhibitor protein (SSTI) from seeds of the medicinal plant Solanum surattense was isolated, purified and characterized. SSTI showed a single band on SDS-PAGE corresponding to 11.4 kDa molecular weight. It is a glycoprotein (2.8% glycosylation) that differentially interacted with trypsin and chymotrypsin in a concentration-dependent manner. Its peptide sequence is similar to other Bowman-Birk type protease inhibitors found in Glycine max and Phaseolus acutifolius. The inhibitory activity was stable over a wide range of pH (1-10) and temperatures (10-100° C). Far-UV Circular Dichroism (CD) studies showed that SSTI contains ß sheets (~ 23%) and α helix (~ 6%) and demonstrated structural stability at wide pH and high temperature. The kinetic analysis revealed a noncompetitive (mixed) type nature of SSTI and low inhibitor constant (Ki) values (16.6 × 10-8 M) suggested strong inhibitory activity. Isothermal titration calorimetric analysis revealed its high affinity towards trypsin with dissociation constant (Kd) 2.28 µM.

Comparative phosphoproteomic analysis unravels MAPK1 regulated phosphoproteins in Leishmania donovani.

Mitogen Activated Protein Kinase1 (MAPK1) of Leishmania donovani functions as key regulators of various cellular activities, which seem to be imperative for parasite survival, infectivity, drug resistance and post-translational modification of chaperones/co-chaperones. However, very less is known about LdMAPK1 target proteins. With recent advancements in proteomics, we aimed to identify phosphoproteins which were differentially expressed in LdMAPK1 overexpressing (Dd8++/++) and single replacement mutants (Dd8+/) as compared to wild type (Dd8+/+) parasites, utilizing LC-MS/MS approach. An in-depth label-free phospoproteomic analysis revealed that modulation of LdMAPK1 expression significantly modulates expression levels of miscellaneous phosphoproteins which may act as its targets/substrates. Out of 1974 quantified phosphoproteins in parasite, 140 were significantly differentially expressed in MAPK1 overexpressing and single replacement mutants. These differentially expressed phosphoproteins are majorly associated with metabolism, signal transduction, replication, transcription, translation, transporters and cytoskeleton/motor proteins, hence suggested that MAPK1 may act in concert to modulate global biological processes. The study further implicated possible role of LdMAPK1 in regulation and management of stress machinery in parasite through post translational modifications. Precisely, comparative phosphoproteomics study has elucidated significant role of LdMAPK1 in regulating various pathways contributing in parasite biology with relevance to future drug development. SIGNIFICANCE: MAPKinase1, the downstream kinase of MAPK signal transduction pathway, has drawn much attention as potential therapeutic drug target due to their indispensable role in survival and infectivity of Leishmania donovani. However, limited information is available about its downstream effector proteins/signaling networks. Utilizing label free LC-MS/MS analysis, phosphoproteome of LdMAPK1 over-expressing (Dd8++/++) and LdMAPK1 single replacement mutants (Dd8+/-) with wild type (Dd8+/+) parasites was compared and identified 140 LdMAPK1 modulated phosphoproteins, mainly involved in pathways like signal transduction, metabolism, transcriptional, translational, post-translational modification and regulation of heat shock proteins. Interestingly, LdMAPK1 interacts directly with only six phosphoproteins i.e. casein kinase, casein kinase II, HSP83/HSP90, LACK, protein kinase and serine/threonine protein kinase. Thus, the study elucidates significant role of LdMAPK1 in Leishmania biology which may drive drug-discovery efforts against visceral leishmaniasis.

Albumin in Advanced Liver Diseases: The Good and Bad of a Drug!

Human serum albumin is the most abundant plasma protein, and it regulates diverse body functions. In patients with advanced and decompensated cirrhosis, serum albumin levels are low because of a reduction in the hepatocyte mass due to disease per se and multiple therapeutic interventions. Because of their oncotic and nononcotic properties, administration of human albumin solutions (HAS) have been found to be beneficial in patients undergoing large-volume paracentesis or who have hepatorenal syndrome or spontaneous bacterial peritonitis. Albumin also improves the functionality of the immune cells and mitigates the severity and risk of infections in advanced cirrhosis. Its long-term administration can modify the course of decompensated cirrhosis patients by reducing the onset of new complications, improving the quality of life, and probably providing survival benefits. There is, however, a need to rationalize the dose, duration, and frequency of albumin therapy in different liver diseases and stages of cirrhosis. In patients with acute-on-chronic liver failure, potentially toxic oxidized isoforms of albumin increase substantially, especially human nonmercaptalbumin and 2, and nitrosoalbumin. The role of administration of HAS in such patients is unclear. Determining whether removal of the pathological and dysfunctional albumin forms in these patients by "albumin dialysis" is helpful, requires additional studies. Use of albumin is not without adverse events. These mainly include allergic and transfusion reactions, volume overload, antibody formation and coagulation derangements. Considering their cost, limited availability, need for a health care setting for their administration, and potential adverse effects, judicious use of HAS in liver diseases is advocated. There is a need for new albumin molecules and economic alternatives in hepatologic practice.